Prebiotic Oral Care Compositions Containing Amino Acids

ABSTRACT

The present invention provides an oral care composition comprising: (a) a dipeptide of the formula Xaa1-Xaa2 or Xaa2-Xaa1; wherein Xaa1 is an amino acid with a polar uncharged side chain; and Xaa2 is selected from an amino acid with a hydrophobic side chain, an amino acid with a polar uncharged side chain, and proline; or (b) a dipeptide of the formula Xaa3-Xaa4 or Xaa4-Xaa3; wherein Xaa3 is an amino acid with a hydrophobic side chain; and Xaa4 is selected from an amino acid with a hydrophobic side chain and an amino acid with a charged side chain.

BACKGROUND

The oral cavity contains many different species of bacteria. Somespecies of oral pathogenic bacteria (e.g. Porphyromonas gingivalis,Tannerella forsythia and Aggregatibacter actinomycetemcomitans) havebeen implicated in the development of periodontal diseases, such asperiodontitis, gingivitis, necrotizing periodontitis, necrotizinggingivitis and peri-implantitis. Certain species of oral pathogenicbacteria have been implicated in tooth decay (e.g. Streptococcusmutans).

It is believed that certain species of oral bacteria are beneficial formaintaining the health of the periodontium. Without being bound by anytheory, it is believed that these beneficial oral bacteria can interferewith colonization by pathogenic oral bacteria of the oral epithelium.For example, studies have shown that Streptococcus oralis, Streptococcusmitis and Streptococcus salivarius have inhibitory effects on A.actinomycetemcomitans colonization of epithelial cells in vitro (W.Teughels et al., J Dent Res 86(7), 611-617, 2007). It has also beenshown, using a canine model, that the application of beneficial bacteriato periodontal pockets following root planing delays and reducesrecolonization of the periodontal pockets by pathogenic bacteria (W.Teughels, et al., J Dent Res, 86(11), 1078-1082, 2007). The beneficialbacteria Streptococcus oralis, Streptococcus mitis and Streptococcussalivarius have also been shown to inhibit A.actinomycetemcomitans-induced production of the inflammatory cytokineinterleukin-8 (IL-8) by the human oral keratinocyte cell line HOK-18A,which inflammatory response is implicated in periodontitis-relatedtissue destruction (I. Sliepen et al., J Dent Res 88(11), 1026-1030,2009).

It is believed that selective stimulation of beneficial oral bacteriamay provide a valid preventative approach for oral health, for examplein the prevention of periodontitis.

BRIEF SUMMARY

In one aspect, the present invention provides an oral care compositioncomprising: (a) a dipeptide of the formula Xaa1-Xaa2 or Xaa2-Xaa1;wherein Xaa1 is an amino acid with a polar uncharged side chain; andXaa2 is selected from an amino acid with a hydrophobic side chain, anamino acid with a polar uncharged side chain, and proline; or (b) adipeptide of the formula Xaa3-Xaa4 or Xaa4-Xaa3; wherein Xaa3 is anamino acid with a hydrophobic side chain; and Xaa4 is selected from anamino acid with a hydrophobic side chain and an amino acid with acharged side chain.

Optionally, Xaa1, Xaa2, Xaa3 and Xaa4 are L-amino acids.

Optionally, the dipeptide is present in the composition in an amount offrom 0.01 weight % to 10 weight %, based on the weight of the oral carecomposition.

Optionally, Xaa2 is an amino acid with an aliphatic hydrophobic sidechain. Further optionally, Xaa2 is alanine, isoleucine, leucine orvaline. Still further optionally, Xaa2 is leucine or valine.

Alternatively, Xaa2 is asparagine, cysteine, glutamine, methionine,serine or threonine. Optionally, Xaa2 is asparagine or glutamine.

Alternatively, Xaa2 is proline.

Optionally, Xaa1 is serine, methionine, threonine, cysteine, asparagineor glutamine. Further optionally, Xaa1 is serine. Alternatively, Xaa1 ismethionine.

Optionally, Xaa3 is an amino acid with an aromatic hydrophobic sidechain. Further optionally, Xaa3 is tryptophan, phenylalanine, ortyrosine. Still further optionally, Xaa3 is tryptophan. Alternatively,Xaa3 is phenylalanine.

Optionally, Xaa4 is aspartic acid, glutamic acid, arginine, histidine orlysine. Further optionally, Xaa4 is glutamic acid or histidine.

Alternatively, Xaa4 is an amino acid with an aliphatic hydrophobic sidechain. Optionally, Xaa4 is alanine, isoleucine, leucine or valine.

Optionally, the composition is a dentifrice, a toothpaste, a gel, atooth powder, a mouthwash, a mouthrinse, a lozenge, a tablet, a spray, agum, or a film.

Optionally, the composition further comprises at least one species ofbacteria that has beneficial effects on oral health. Further optionally,the species of bacteria that has beneficial effects on oral health isselected from Streptococcus mitis, Streptococcus salivarius,Streptococcus oxalis, Actinomyces viscosus, Veillonella parvula,Streptococcus gordonii, Capnocytophaga sputigena, Actinomyces naeslundiiand combinations thereof.

In another aspect, the present invention provides an oral carecomposition of the present invention, for use in selectively promoting,in an oral cavity: growth, metabolic activity or colonization ofbacteria that have beneficial effects on oral health, relative togrowth, metabolic activity or colonization of pathogenic oral bacteria.In another aspect, the present invention provides an oral carecomposition of the present invention, for use in selectively promoting,in an oral cavity, biofilm formation by bacteria that have beneficialeffects on oral health, relative to biofilm formation by pathogenic oralbacteria. In another aspect, the present invention provides an oral carecomposition of the present invention, for use in maintaining and/orre-establishing a healthy oral microbiota. In another aspect, thepresent invention provides an oral care composition of the presentinvention, for use in preventing one or more of gingivitis,periodontitis, peri-implantitis, peri-implant mucositis, necrotizinggingivitis, necrotizing periodontitis and caries.

In another aspect, the present invention provides a method ofselectively promoting, in an oral cavity of a subject: growth, metabolicactivity or colonization of bacteria that have beneficial effects onoral health, relative to growth, metabolic activity or colonization ofpathogenic oral bacteria; the method comprising contacting the oralcavity with an oral care composition of the present invention. Inanother aspect, the present invention provides a method of selectivelypromoting, in an oral cavity of a subject, biofilm formation by bacteriathat have beneficial effects on oral health, relative to biofilmformation by pathogenic oral bacteria; the method comprising contactingthe oral cavity with an oral care composition of the present invention.The present invention also provides a method of maintaining and/orre-establishing a healthy oral microbiota in a subject, the methodcomprising contacting an oral cavity of the subject with an oral carecomposition of the present invention. The present invention alsoprovides a method of preventing one or more of gingivitis,periodontitis, peri-implantitis, peri-implant mucositis, necrotizinggingivitis, necrotizing periodontitis and caries in a subject, themethod comprising contacting an oral cavity of the subject with an oralcare composition of the present invention.

In another aspect, the present invention provides the use, in an oralcare composition, of (i) a dipeptide of the formula Xaa1 -Xaa2 orXaa2-Xaa1; wherein Xaa1 is an amino acid with a polar uncharged sidechain; and Xaa2 is selected from an amino acid with a hydrophobic sidechain, an amino acid with a polar uncharged side chain, and proline; or(ii) a dipeptide of the formula Xaa3-Xaa4 or Xaa4-Xaa3; wherein Xaa3 isan amino acid with a hydrophobic side chain; and Xaa4 is selected froman amino acid with a hydrophobic side chain and an amino acid with acharged side chain; to: (a) selectively promote growth, metabolicactivity or colonization of bacteria that have beneficial effects onoral health, relative to growth, metabolic activity or colonization ofpathogenic oral bacteria; (b) selectively promote biofilm formation bybacteria that have beneficial effects on oral health, relative tobiofilm formation by pathogenic oral bacteria; (c) maintain and/orre-establish a healthy oral microbiota in a subject; or (d) prevent oneor more of gingivitis, periodontitis, peri-implantitis, peri-implantmucositis, necrotizing gingivitis, necrotizing periodontitis and cariesin a subject.

Optionally, the dipeptide selectively promotes growth, metabolicactivity or colonization of bacteria that have beneficial effects onoral health, relative to growth, metabolic activity or colonization ofpathogenic oral bacteria, after 24 hours incubation with the bacteriathat have beneficial effects on oral health and the pathogenic oralbacteria.

Optionally, the dipeptide selectively promotes growth, metabolicactivity or colonization of bacteria that have beneficial effects onoral health, relative to growth, metabolic activity or colonization ofpathogenic oral bacteria, after 48 hours incubation with the bacteriathat have beneficial effects on oral health and the pathogenic oralbacteria.

Further areas of applicability of the present invention will becomeapparent from the detailed description provided hereinafter. It shouldbe understood that the detailed description and specific examples, whileindicating the preferred embodiment of the invention, are intended forpurposes of illustration only and are not intended to limit the scope ofthe invention.

DETAILED DESCRIPTION

The following description of the preferred embodiment(s) is merelyexemplary in nature and is in no way intended to limit the invention,its application, or uses.

As used throughout, ranges are used as shorthand for describing each andevery value that is within the range. Any value within the range can beselected as the terminus of the range. In addition, all references citedherein are hereby incorporated by referenced in their entireties. In theevent of a conflict in a definition in the present disclosure and thatof a cited reference, the present disclosure controls.

Unless otherwise specified, all percentages and amounts expressed hereinand elsewhere in the specification should be understood to refer topercentages by weight. The amounts given are based on the active weightof the material.

As used herein, the expression “oral cavity” includes not only thecavity itself but also the saliva, teeth, gingiva, periodontal pockets,cheeks, tongue, mucosa, tonsils, any implants, and any device orstructure which is placed into the oral cavity. The tonsils provide areservoir (tonsil stones) for growth of anaerobic bacteria which maygenerate bad breath.

As used herein, the phrase “a healthy oral microbiota” refers to themicrobial population of the oral cavity when the oral cavity is in anon-diseased state (for example, when there is no periodontal diseasepresent, e.g. gingivitis, periodontitis, caries, peri-implantitis,peri-implant mucositis, necrotizing gingivitis and/or necrotizingperiodontitis), i.e. a health-associated oral microbiota.

The term “beneficial bacteria” encompasses those bacteria which arepresent in the oral cavity in higher numbers or proportions in a healthyoral cavity, but which are present in lower numbers or proportions inconditions of oral disease (such as, for example, gingivitis,periodontitis, caries, peri-implantitis, peri-implant mucositis,necrotizing gingivitis and necrotizing periodontitis). This term alsoincludes bacteria from oral or non-oral origins which have provenbeneficial effects on oral health by preventing or treating oraldiseases, which may be already present in the oral cavity or may beintentionally introduced into the oral cavity (for example asprobiotics). Beneficial bacteria may, by their presence or metabolicactivity, result in: lowering the number or proportion of pathogenicoral bacteria; lowering inflammation and inflammatory processes;lowering the metabolic activity of pathogenic species; lowering theproduction or inhibiting virulence factors produced by pathogenicbacteria; lowering or inhibiting biofilm formation; occupying a nichewhich may otherwise be colonized by pathogens; limiting a pathogen'sability to adhere to oral surfaces; affecting the viability, metabolicactivity or growth of a pathogen; lowering the ability of a pathogen toproduce virulence factors; degrading virulence factors produced by thepathogen or the oral microbiota; and/or attenuating the host response topathogenic species. As discussed above, it is believed that thesebeneficial oral bacteria can interfere with colonization of the oralepithelium by pathogenic oral bacteria. It is believed that selectivestimulation of beneficial oral bacteria may provide a valid preventativeapproach for oral health, for example in the prevention ofperiodontitis.

Bacteria require certain substrates in order to enable them to grow,multiply, be metabolically active and to colonize. Certain substratescan be used selectively by certain microorganisms (e.g. bacteria) tofavor their growth, metabolic activity, multiplication and colonization,and thereby directly or indirectly suppress the growth of othermicroorganisms. The present inventors investigated the possibility ofmodifying the oral microbiota of a subject by selectively increasing thesubject's indigenous beneficial oral bacterial population, by providingthe beneficial oral bacteria with appropriate substrates forgrowth/multiplication.

Examples of beneficial bacteria include Streptococcus mitis,Streptococcus salivarius, Streptococcus oxalis, Actinomyces viscosus,Veillonella parvula, Streptococcus gordonii, Capnocytophaga sputigenaand Actinomyces naeslundii. Examples of pathogenic bacteria includeStreptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis,Fusobacterium nucleatum, Tannerella forsythia, Aggregatibacteractinomycetemcomitans and Streptococcus sobrinus.

The present inventors have found that certain dipeptides are able tocause an increase in growth, metabolic activity and biofilm formation ofbeneficial oral bacteria, while not causing an increase in growth,metabolic activity and biofilm formation of pathogenic oral bacteria.

The present invention therefore provides an oral care compositioncomprising: (a) a dipeptide of the formula Xaa1-Xaa2 or Xaa2-Xaa1;wherein Xaa1 is an amino acid with a polar uncharged side chain; andXaa2 is selected from an amino acid with a hydrophobic side chain, anamino acid with a polar uncharged side chain, and proline; or (b) adipeptide of the formula Xaa3-Xaa4 or Xaa4-Xaa3; wherein Xaa3 is anamino acid with a hydrophobic side chain; and Xaa4 is selected from anamino acid with a hydrophobic side chain and an amino acid with acharged side chain.

In some embodiments, Xaa1, Xaa2, Xaa3 and Xaa4 are L-amino acids.

In some embodiments, the amino acid Xaa1 (an amino acid with a polaruncharged side chain) is serine (Ser), methionine (Met), threonine(Thr), cysteine (Cys), asparagine (Asn) or glutamine (Gln). In certainembodiments, Xaa1 is serine or threonine. In other embodiments, Xaa1 ismethionine or cysteine. In some embodiments, Xaa1 is serine. In otherembodiments, Xaa1 is methionine.

When Xaa2 is an amino acid with a hydrophobic side chain, Xaa2 may incertain embodiments be an amino acid with an aliphatic hydrophobic sidechain. In some of these embodiments, Xaa2 is alanine (Ala), isoleucine(Ile), leucine (Leu) or valine (Val). In certain embodiments, Xaa2 isleucine or valine. In certain embodiments, the dipeptide is Ser-Leu,Ser-Val, Leu-Ser or Val-Ser. In some embodiments, the dipeptide isSer-Leu or Ser-Val.

When Xaa2 is an amino acid with a polar uncharged side chain, Xaa2 mayin certain embodiments be asparagine (Asn), cysteine (Cys), glutamine(Gln), methionine (Met), serine (Ser) or threonine (Thr). In some ofthese embodiments, Xaa2 is asparagine or glutamine. In otherembodiments, Xaa2 is cysteine or methionine. In other embodiments, Xaa2is serine or threonine. In certain embodiments, the dipeptide isSer-Asn, Ser-Gln, Asn-Ser or Gln-Ser. In some embodiments, the dipeptideis Ser-Asn or Ser-Gln.

In some embodiments, Xaa2 is proline (Pro). In one embodiment thedipeptide is Met-Pro.

In some embodiments, the amino acid Xaa3 (an amino acid with ahydrophobic side chain) is an amino acid with an aromatic hydrophobicside chain. In certain embodiments, Xaa3 is tryptophan (Trp),phenylalanine (Phe), or tyrosine (Tyr). In some embodiments, Xaa3 istryptophan. In other embodiments, Xaa3 is phenylalanine.

When Xaa4 is an amino acid with a hydrophobic side chain, Xaa4 may incertain embodiments be an amino acid with an aliphatic hydrophobic sidechain. In some of these embodiments, Xaa4 is alanine (Ala), isoleucine(Ile), leucine (Leu) or valine (Val). In certain embodiments, Xaa4 isleucine or valine. In certain embodiments, Xaa4 is leucine. In certainembodiments, the dipeptide is Trp-Leu or Leu-Trp.

When Xaa4 is an amino acid with a charged side chain, Xaa4 may incertain embodiments be aspartic acid (Asp), glutamic acid (Glu),arginine (Arg), histidine (His) or lysine (Lys). In certain embodiments,Xaa4 is aspartic acid or glutamic acid. In other embodiments, Xaa4 isarginine, histidine or lysine. In other embodiments, Xaa4 is glutamicacid or histidine. In some embodiments, the dipeptide is Trp-Glu,Trp-His, Glu-Trp, Glu-His, Phe-Glu or Glu-Phe. In some embodiments, thedipeptide is Glu-Trp, His-Trp or Phe-Glu. In some embodiments, thedipeptide is Phe-Glu.

In some embodiments, the dipeptide is Ser-Leu, Ser-Val, Ser-Asn,Ser-Gln, Met-Pro, Phe-Glu, Glu-Trp, Trp-Leu or His-Trp. In someembodiments, the dipeptide is Ser-Leu or Ser-Val. In other embodiments,the dipeptide is Ser-Asn, Ser-Gln, Met-Pro, Phe-Glu, Glu-Trp, Trp-Leu orHis-Trp.

In some embodiments, the dipeptide is present in the composition in anamount of from 0.01 weight % to 10 weight %, from 0.05 weight % to 7.5weight %, from 0.1 weight % to 5 weight %, or from 1 weight % to 2.5weight %, based on the weight of the oral care composition.

In any of the above embodiments, the dipeptide may be present in thecomposition in an amount of from 1 μmol/ml to 50 μmol/ml, from 2.5μmol/ml to 35 μmol/ml, from 5 μmol/ml to 25 μmol/ml, or from 10 μmol/m1to 20 μmol/ml. In certain embodiments, the dipeptide is present in thecomposition in an amount of 5 μmol/ml to 10 μmol/ml, or from 10 μmol/mlto 25 μmol/ml.

The oral care composition as described in any of the above embodimentsmay be a dentifrice, a toothpaste, a gel, a tooth powder, a mouthwash, amouthrinse, a lozenge (which may be dissolvable or chewable), a tablet,a spray, a gum, or a film (which may be wholly or partially dissolvable,or indissolvable).

The oral care compositions as described in any of the above embodimentsmay further comprise at least one species of bacteria that hasbeneficial effects on oral health. In these embodiments, the compositionmay be referred to as a synbiotic composition, as it contains both aprobiotic (the bacteria which has beneficial effects on oral health) anda prebiotic (the dipeptide which stimulates the growth, metabolicactivity and/or colonization of the probiotic bacteria). In certainembodiments, the species of bacteria that has beneficial effects on oralhealth is selected from Streptococcus mitis, Streptococcus salivarius,Streptococcus oxalis, Actinomyces viscosus, Veillonella parvula,Streptococcus gordonii, Capnocytophaga sputigena, Actinomyces naeslundiiand combinations thereof.

The present invention also provides an oral care composition accordingto any of the above-described embodiments, for use in (a) selectivelypromoting, in an oral cavity: growth, metabolic activity or colonizationof bacteria that have beneficial effects on oral health, relative togrowth, metabolic activity or colonization of pathogenic oral bacteria;(b) selectively promoting, in an oral cavity, biofilm formation bybacteria that have beneficial effects on oral health, relative tobiofilm formation by pathogenic oral bacteria; (c) maintaining and/orre-establishing a healthy oral microbiota; (d) preventing one or more ofgingivitis, periodontitis, peri-implantitis, peri-implant mucositis,necrotizing gingivitis, necrotizing periodontitis and caries.

The present invention also provides for the use, in an oral carecomposition, of a dipeptide as described in any of the above embodimentsto (a) selectively promote growth, metabolic activity or colonization ofbacteria that have beneficial effects on oral health, relative togrowth, metabolic activity or colonization of pathogenic oral bacteria;(b) selectively promote biofilm formation by bacteria that havebeneficial effects on oral health, relative to biofilm formation bypathogenic oral bacteria; (c) maintain and/or re-establish a healthyoral microbiota in a subject; or (d) prevent one or more of gingivitis,periodontitis, peri-implantitis, peri-implant mucositis, necrotizinggingivitis, necrotizing periodontitis and caries in a subject.

The present invention also provides a method of (a) selectivelypromoting, in an oral cavity of a subject: growth, metabolic activity orcolonization of bacteria that have beneficial effects on oral health,relative to growth, metabolic activity or colonization of pathogenicoral bacteria; (b) selectively promoting, in an oral cavity of asubject, biofilm formation by bacteria that have beneficial effects onoral health, relative to biofilm formation by pathogenic oral bacteria;(c) maintaining and/or re-establishing a healthy oral microbiota in asubject; or (d) preventing one or more of gingivitis, periodontitis,peri-implantitis, peri-implant mucositis, necrotizing gingivitis,necrotizing periodontitis and caries in a subject. Each of these methodscomprises contacting an oral cavity of the subject with an oral carecomposition according to any of the above-described embodiments.

In the above methods, the subject may be a mammal. In some embodiments,the subject is a human. In some embodiments, the subject is an animal,for example a companion animal (e.g. a cat or dog).

In certain embodiments, the contacting of the oral cavity with the oralcare composition comprises applying the oral care composition to theoral cavity using a brush, rinsing the oral cavity with the oral carecomposition in the form of a mouthwash, or spraying the oral carecomposition into the oral cavity using, for example, an atomizer.

In any of the above embodiments of the methods and uses, the bacteriathat have beneficial effects of oral health may be selected fromStreptococcus mitis, Streptococcus salivarius, Streptococcus oralis,Actinomyces viscosus, Veillonella parvula, Streptococcus gordonii,Capnocytophaga sputigena, Actinomyces naeslundii and combinationsthereof. Additionally or alternatively, the pathogenic bacteria may beselected from Streptococcus mutans, Prevotella intermedia, Porphyromonasgingivalis, Fusobacterium nucleatum, Tannerella forsythia,Aggregatibacter actinomycetemcomitans, Streptococcus sobrinus andcombinations thereof.

In any of the above embodiments wherein the methods and uses concern theselective promotion (in an oral cavity of a subject) of growth,metabolic activity or colonization of bacteria that have beneficialeffects on oral health, relative to growth, metabolic activity orcolonization of pathogenic oral bacteria, the bacteria that havebeneficial effects of oral health may be selected from Streptococcusmitis, Streptococcus salivarius, Actinomyces viscosus, Veillonellaparvula, Capnocytophaga sputigena and combinations thereof. Additionallyor alternatively, the pathogenic bacteria may be selected fromStreptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis,Fusobacterium nucleatum, Tannerella forsythia, Aggregatibacteractinomycetemcomitans, Streptococcus sobrinus and combinations thereof,and in certain embodiments may be selected from Prevotella intermedia,Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythiaand combinations thereof.

In any of the above embodiments wherein the methods and uses concern theselective promotion of biofilm formation by bacteria that havebeneficial effects on oral health, relative to biofilm formation bypathogenic oral bacteria, the pathogenic bacteria may be selected fromStreptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis,Fusobacterium nucleatum, Tannerella forsythia, Aggregatibacteractinomycetemcomitans and combinations thereof. Additionally oralternatively, the bacteria that have beneficial effects of oral healthmay be selected from Streptococcus oralis, Streptococcus gordonii andcombinations thereof.

In some embodiments, the dipeptide selectively promotes growth,metabolic activity or colonization of bacteria that have beneficialeffects on oral health, relative to growth, metabolic activity orcolonization of pathogenic oral bacteria, after 24 hours incubation withthe bacteria that have beneficial effects on oral health and thepathogenic oral bacteria.

In some embodiments, the dipeptide selectively promotes growth,metabolic activity or colonization of bacteria that have beneficialeffects on oral health, relative to growth, metabolic activity orcolonization of pathogenic oral bacteria, after 48 hours incubation withthe bacteria that have beneficial effects on oral health and thepathogenic oral bacteria.

In the compositions of the present invention comprising dipeptides, thesequence of the amino acids of the dipeptide is reported from theN-terminal end (containing the free amino group) to the C-terminal end(containing the free carboxyl group). Therefore, for the example of“Ser-Val”, the structure is as shown in Formula I; and, for “His-Trp”,the structure is as shown in Formula II:

The oral care compositions of the present invention may further compriseadditional ingredients. These additional ingredients may include, butare not limited to, abrasives, diluents, bicarbonate salts, pH modifyingagents, surfactants, foam modulators, thickening agents, humectants,sweeteners, flavorants, pigments, antibacterial agents, anticariesagents, anticalculus or tartar control agents, and mixtures thereof.

In some embodiments, particularly (but not limited to) thoseembodiments, wherein the oral care composition is a toothpaste, thecompositions of the present invention may further comprise an abrasive.Abrasives that may be used include silica abrasives such as precipitatedor hydrated silicas having a mean particle size of up to about 20microns, such as Zeodent 105 and Zeodent 114 marketed by J.M. HuberChemicals Division, Havre de Grace, Md. 21078, or Sylodent 783 marketedby Davison Chemical Division of W.R. Grace & Company. Abrasives such asSorbosil AC 43 from PQ Corporation may also be included. Other usefuldentifrice abrasives include aluminium oxide, aluminum silicate,calcined alumina, bentonite or other siliceous materials, insolublephosphates, and mixtures thereof. The abrasive may be present in anamount of from 5 to 30 wt % based on the weight of the composition,optionally from 10 to 20 wt % based on the weight of the composition. Incertain embodiments, particularly (but not limited to) those embodimentswherein the oral care composition is a mouthwash or mouthrinse, thecompositions may be free of abrasives.

In some embodiments, the oral care compositions of the present inventioncomprise at least one bicarbonate salt useful for example to impart a“clean feel” to teeth and gums due to effervescence and release ofcarbon dioxide. Any orally acceptable bicarbonate can be used, includingwithout limitation, alkali metal bicarbonates such as sodium andpotassium bicarbonates, ammonium bicarbonate and the like. The one ormore additional bicarbonate salts are optionally present in a totalamount of about 0.1 wt. % to about 50 wt. %, for example about 1 wt. %to 20 wt. %, by total weight of the composition.

In some embodiments, the oral care compositions of the present inventioncomprise at least one pH modifying agent. Such agents include acidifyingagents to lower pH, basifying agents to raise pH, and buffering agentsto control pH within a desired range. For example, one or more compoundsselected from acidifying, basifying and buffering agents can be includedto provide a pH of 2 to 10, or in various illustrative embodiments, 2 to8, 3 to 9, 4 to 8, 5 to 7, 6 to 10, 7 to 9, etc. Any orally acceptablepH modifying agent can be used, including without limitation,carboxylic, phosphoric and sulfonic acids, acid salts (e.g., monosodiumcitrate, disodium citrate, monosodium malate, etc.), alkali metalhydroxides such as sodium hydroxide, carbonates such as sodiumcarbonate, bicarbonates, sesquicarbonates, borates, silicates,phosphates (e.g., monosodium phosphate, trisodium phosphate), imidazoleand the like. One or more pH modifying agents are optionally present ina total amount effective to maintain the composition in an orallyacceptable pH range.

The oral care compositions of the invention may also comprise at leastone surfactant. Any orally acceptable surfactant, most of which areanionic, nonionic or amphoteric, can be used. Suitable anionicsurfactants include without limitation, water-soluble salts of C₈₋₂₀alkyl sulfates, sulfonated monoglycerides of C₈₋₂₀ fatty acids,sarcosinates, taurates and the like. Illustrative examples of these andother classes include sodium lauryl sulfate, sodium coconutmonoglyceride sulfonate, sodium lauryl sarcosinate, sodium laurylisoethionate, sodium laureth carboxylate and sodium dodecylbenzenesulfonate. Suitable nonionic surfactants include withoutlimitation, poloxamers, polyoxyethylene sorbitan esters, fatty alcoholethoxylates, alkylphenol ethoxylates, tertiary amine oxides, tertiaryphosphine oxides, dialkyl sulfoxides and the like. Suitable amphotericsurfactants include without limitation, derivatives of C₈₋₂₀ aliphaticsecondary and tertiary amines having an anionic group such ascarboxylate, sulfate, sulfonate, phosphate or phosphonate. Betaines mayalso be used, a suitable example of which is cocoamidopropyl betaine.One or more surfactants are optionally present in a total amount ofabout 0.01 wt. % to about 10 wt. %, for example, from about 0.05 wt. %to about 5 wt. %, or from about 0.1 wt. % to about 2 wt. % by totalweight of the composition.

The oral care compositions of the invention may comprise at least onefoam modulator, useful for example to increase amount, thickness orstability of foam generated by the composition upon agitation. Anyorally acceptable foam modulator can be used, including withoutlimitation, polyethylene glycols (PEGs), also known as polyoxyethylenes.High molecular weight PEGs are suitable, including those having anaverage molecular weight of 200,000 to 7,000,000, for example 500,000 to5,000,000, or 1,000,000 to 2,500,000. One or more PEGs are optionallypresent in a total amount of about 0.1 wt. % to about 10 wt. %, forexample from about 0.2 wt. % to about 5 wt. %, or from about 0.25 wt. %to about 2 wt. %, by total weight of the composition.

The oral care compositions of the present invention may comprise atleast one thickening agent, useful for example to impart a desiredconsistency and/or mouth feel to the composition. Any orally acceptablethickening agent can be used, including without limitation, carbomers,also known as carboxyvinyl polymers, carrageenans, also known as Irishmoss and more particularly ι-carrageenan (iota-carrageenan), cellulosicpolymers such as hydroxyethylcellulose, carboxymethylcellulose (CMC) andsalts thereof, e.g., CMC sodium, natural gums such as karaya, xanthan,gum arabic and tragacanth, colloidal magnesium aluminum silicate,colloidal silica and the like. A preferred class of thickening orgelling agents includes a class of homopolymers of acrylic acidcrosslinked with an alkyl ether of pentaerythritol or an alkyl ether ofsucrose, or carbomers. Carbomers are commercially available from B. F.Goodrich as the Carbopol® series. Particularly preferred Carbopolsinclude Carbopol 934, 940, 941, 956, 974P, and mixtures thereof. Silicathickeners such as DT 267 (from PPG Industries) may also be used. One ormore thickening agents are optionally present in a total amount of fromabout 0.01 wt. % to 15 wt. %, for example from about 0.1 wt. % to about10 wt. %, or from about 0.2 wt. % to about 5 wt. %, by total weight ofthe composition.

The compositions of the invention may comprise at least one viscositymodifier, useful for example to help inhibit settling or separation ofingredients or to promote re-dispersibility upon agitation of a liquidcomposition. Any orally acceptable viscosity modifier can be used,including without limitation, mineral oil, petrolatum, clays andorganomodified clays, silica and the like. One or more viscositymodifiers are optionally present in a total amount of from about 0.01wt. % to about 10 wt. %, for example, from about 0.1 wt. % to about 5wt. %, by total weight of the composition.

The compositions of the invention may also comprise at least onehumectant. Any orally acceptable humectant can be used, includingwithout limitation, polyhydric alcohols such as glycerin, sorbitol(optionally as a 70 wt. % solution in water), xylitol or low molecularweight polyethylene glycols (PEGs). Most humectants also function assweeteners. One or more humectants are optionally present in a totalamount of from about 1 wt. % to about 70 wt. %, for example, from about1 wt.% to about 50 wt.%, from about 2 wt.% to about 25 wt.%, or fromabout 5 wt. % to about 15 wt. %, by total weight of the composition.

The oral care compositions of the present invention may comprise atleast one sweetener, useful for example to enhance taste of thecomposition. One or more sweeteners are optionally present in a totalamount depending strongly on the particular sweetener(s) selected, buttypically 0.005 wt. % to 5 wt. %, by total weight of the composition,optionally 0.005 wt. % to 0.2 wt. %, further optionally 0.05 wt. % to0.1 wt. % by total weight of the composition.

The compositions of the present invention may also comprise at least oneflavorant, useful for example to enhance taste of the composition. Anyorally acceptable natural or synthetic flavorant can be used, includingwithout limitation tea flavours, vanillin, sage, marjoram, parsley oil,spearmint oil, cinnamon oil, oil of wintergreen (methylsalicylate),peppermint oil, clove oil, bay oil, anise oil, eucalyptus oil, citrusoils, fruit oils and essences including those derived from lemon,orange, lime, grapefruit, apricot, banana, grape, apple, strawberry,cherry, pineapple, etc., bean- and nut-derived flavors such as coffee,cocoa, cola, peanut, almond, etc., adsorbed and encapsulated flavorantsand the like. Also encompassed within flavorants herein are ingredientsthat provide fragrance and/or other sensory effect in the mouth,including cooling or warming effects. Such ingredients illustrativelyinclude menthol, menthyl acetate, menthyl lactate, camphor, eucalyptusoil, eucalyptol, anethole, eugenol, cassia, oxanone, α-irisone, propenylguaiethol, thymol, linalool, benzaldehyde, cinnamaldehyde,N-ethyl-p-menthan-3 -carboxamine, N,2,3-trimethyl-2-isopropylbutanamide,3 -(1-menthoxy)-propane-1,2-diol, cinnamaldehyde glycerol acetal (CGA),menthone glycerol acetal (MGA) and the like. One or more flavorants areoptionally present in a total amount of from about 0.01 wt. % to about 5wt. %, for example, from about 0.03 wt. % to about 2.5 wt. %, optionallyabout 0.05 wt. % to about 1.5 wt. %, further optionally about 0.1 wt. %to about 0.3 wt. % by total weight of the composition.

The compositions of the invention may comprise at least one colorant.Colorants herein include pigments, dyes, lakes and agents imparting aparticular luster or reflectivity such as pearling agents. Any orallyacceptable colorant can be used, including without limitation talc,mica, magnesium carbonate, calcium carbonate, magnesium silicate,magnesium aluminum silicate, silica, titanium dioxide, zinc oxide, red,yellow, brown and black iron oxides, ferric ammonium ferrocyanide,manganese violet, ultramarine, titaniated mica, bismuth oxychloride, andthe like. One or more colorants are optionally present in a total amountof from about 0.001 wt. % to about 20 wt. %, for example, from about0.01 wt. % to about 10 wt. %, or from about 0.1 wt. % to about 5 wt. %,by total weight of the composition.

The compositions of the present invention may also comprise anantibacterial or preservative agent, such as chlorhexidine, triclosan,quaternary ammonium compounds (for example benzalkonium chloride) orparabens such as methylparaben or propylparaben. One or moreantibacterial or preservative agent is optionally present in thecomposition in a total amount of from about 0.01 wt. % to about 0.5 wt.%, optionally about 0.05 wt. % to about 0.1 wt. % by total weight of thecomposition.

The oral care compositions may also comprise a fluoride ion source.Fluoride ion sources include, but are not limited to: stannous fluoride,sodium fluoride, potassium fluoride, potassium monofluorophosphate,sodium monofluorophosphate, ammonium monofluorophosphate, sodiumfluorosilicate, ammonium fluorosilicate, amine fluoride such as olaflur(N′-octadecyltrimethylendiamine-N,N,N′-tris(2-ethanol)-dihydrofluoride),ammonium fluoride, and combinations thereof. In certain embodiments thefluoride ion source includes stannous fluoride, sodium fluoride, aminefluorides, sodium monofluorophosphate, as well as mixtures thereof. Incertain embodiments, the oral care composition of the invention may alsocontain a source of fluoride ions or fluorine-providing ingredient inamounts sufficient to supply about 50 to about 5000 ppm fluoride ion,e.g., from about 100 to about 1000, from about 200 to about 500, orabout 250 ppm fluoride ion. Fluoride ion sources may be added to thecompositions of the invention at a level of about 0.001 wt. % to about10 wt. %, e.g., from about 0.003 wt. % to about 5 wt. %, 0.01 wt. % toabout 1 wt., or about 0.05 wt. %. However, it is to be understood thatthe weights of fluoride salts to provide the appropriate level offluoride ion will obviously vary based on the weight of the counter ionin the salt, and one of skill in the art may readily determine suchamounts. A preferred fluoride salt may be sodium fluoride.

The compositions of the present invention may comprise a salivastimulating agent useful, for example, in amelioration of dry mouth. Anyorally acceptable saliva stimulating agent can be used, includingwithout limitation food acids such as citric, lactic, malic, succinic,ascorbic, adipic, fumaric and tartaric acids, and mixtures thereof. Oneor more saliva stimulating agents are optionally present in salivastimulating effective total amount.

The compositions of the present invention may include antisensitivityagents, e.g., potassium salts such as potassium nitrate, potassiumbicarbonate, potassium chloride, potassium citrate, and potassiumoxalate; capsaicin; eugenol; strontium salts; chloride salts andcombinations thereof. Such agents may be added in effective amounts,e.g., from about 1 wt. % to about 20 wt. % by weight based on the totalweight of the composition, depending on the agent chosen.

The composition of the invention may further comprise an antioxidant.Any orally acceptable antioxidant can be used, including butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), vitamin A,carotenoids, vitamin E, flavonoids, polyphenols, ascorbic acid, herbalantioxidants, chlorophyll, melatonin, and mixtures thereof.

The compositions of the present invention may additionally optionallycomprise a tartar control (anticalculus) agent as provided below. Tartarcontrol agents among those useful herein include salts of the specifiedagents, including alkali metal and ammonium salts. The agents include:phosphates and polyphosphates, polyaminopropanesulfonic acid (AMPS),polyolefin sulfonates, polyolefin phosphates, diphosphonates such asazacycloalkane-2,2-diphosphonates (e.g.,azacycloheptane-2,2-diphosphonic acid), N-methylazacyclopentane-2,3-diphosphonic acid, ethane-1-hydroxy-1,1-diphosphonicacid (EHDP) and ethane-1-amino-1,1-diphosphonate, phosphonoalkanecarboxylic acids and. Useful inorganic phosphate and polyphosphate saltsinclude monobasic, dibasic and tribasic sodium phosphates, sodiumtripolyphosphate, tetrapolyphosphate, sodium trimetaphosphate, sodiumhexametaphosphate and mixtures thereof. Other useful tartar controlagents include polycarboxylate polymers and polyvinyl methylether/maleic anhydride (PVM/MA) copolymers, such as GANTREZ®.

EXAMPLES Example 1

The metabolic activity of various beneficial oral bacteria andpathogenic oral bacteria in the presence of the dipeptides Ser-Leu,Ser-Val, Ser-Asn, Ser-Gln, Met-Pro, Phe-Glu, Glu-Trp, Trp-Leu or His-Trpwas investigated. In the examples, the amino acid residues present inthe dipeptides are L-amino acid residues.

The beneficial oral bacteria tested were Streptococcus mitis,Streptococcus salivarius, Streptococcus oralis, Actinomyces viscosus,Streptococcus gordonii, Capnocytophaga sputigena, Actinomyces naeslundiiand Veillonella parvula. The pathogenic oral bacteria tested wereStreptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis,Fusobacterium nucleatum, Tannerella forsythia, Aggregatibacteractinomycetemcomitans and Streptococcus sobrinus.

Substrates that were able to increase the metabolic activity of at leastone of the above beneficial bacteria while not or only minimallyincreasing the growth of the pathogenic bacteria are considered to beprebiotic substrates.

The extent of metabolic activity of the tested bacteria in the presenceof the above-mentioned dipeptides after 24 hours and 48 hours wasinvestigated through a high throughput phenotype microarray system (PM)for microbial cells (Biolog Inc.). Each PM can contain up to 95different biochemical compounds (e.g. di-peptides) that may act assubstrates for bacteria as well as one negative control, not containingthe substrates. Microarray plates were used in which the different wellsof each plate were pre-loaded (by the manufacturer, Biolog Inc.) withdifferent di-peptides to be tested. Each bacterial species was testedusing a separate microarray plate. Thus, a single bacterial species wastested with a variety of dipeptides on each particular plate. Themicroarrays are based on redox technology, using cell respiration as auniversal reporter. Active cell respiration results in the reduction ofa tetrazolium dye and in the formation of a strong color—for example,when using the tetrazolium dye “Biolog Redox Dye Mix D”, the colorchanges from transparent/colourless to purple when the dye is reduced.The observation of this color change indicates which of the substratesimprove the metabolic activity and health of the cells.

Each bacterial species was collected from blood agar plates (incubatedfor 48 hours anaerobically at 37° C.) and transferred to an IF-0 Baseinoculation fluid (Biolog Inc.), which is a nutritionally poor medium.The resulting cell suspension was adjusted to a transmittance of 42%(relative to the transmittance of the base inoculation fluid with nobacterial species present) at 492 nm using a BioRad SmartSpec 3000Photometer (the adjustment being effected by adding either furtherinoculation fluid or further bacteria until the transmittance of 42% wasachieved). A 1:5 dilution of this suspension was prepared by mixing 3 mLof the 42% transmittance cell suspension with 15 mL of an inoculationfluid (which inoculation fluid was formed by mixing 11.6 mL sterilewater, 62.5 mL IF-0 base inoculation fluid and 0.9 mL of the tetrazoliumdye “Biolog Redox Dye mix D”), resulting in a final cell density whichwas equal to 85% transmittance using a BioRad SmartSpec 3000 Photometer.680 μL of a 2M sodium succinate/200 μM ferric citrate solution indistilled water was added to 68 mL of the 85% transmittance cellsuspension and 100 μL of this mixture was added to each well (thevarious di-peptides being already present in powder form in the relevantwells of the plate as obtained from the supplier i.e. Biolog Inc.).

All plates were incubated in an anaerobic atmosphere at 37° C. Colorchanges were measured spectrophotometrically at 492 nm (ThermoScientific Multiskan Ascent) at 24 hrs and 48 hrs using the same plateat both time points. For each bacterium tested, a respective control wasalso used, the control being a well of the plate which contained theparticular bacterium (cell suspension) but did not contain anydipeptide. After 24 hrs and 48 hrs under continuous shaking in aMultiscan microplate reader, the optical density (OD) of the syntheticmedium at 24 hours and 48 hours was measured (using the microplatereader) at a wavelength of 492 nm for each combination ofbacterium/dipeptide, and for each of their respective controls (i.e.which contained the particular bacterium but no dipeptide). For eachbacterium, the OD value obtained at 24 hrs and at 48 hrs was divided bythe OD value obtained for the respective control at 24 hrs and 48 hrs(respectively), so that the control had a value of 1. A value greaterthan 1 for a particular combination of bacterium with dipeptidetherefore indicates that the increase in metabolic activity of thebacteria after 24 hrs or 48 hrs was greater than the increase observedfor the control.

The experiment was carried out three times for each combination ofbacterium with dipeptide, with each repeat being carried out on adifferent day (thus providing three biological replicas). The controlswere also carried out three times, as above. The values shown are theaverage (mean) of the three single values obtained (as detailed above)for each combination of bacterium with dipeptide, at 24 hrs and at 48hrs. The results are shown in Tables 1 to 4, below:

TABLE 1 Pathogenic bacteria at 24 hrs A actino** F nucleatu P gingivalisP intermedi T forsythia S mutans S sobrinus Ser-Leu 1.6871 1.4813 1.46261.5403 1.3950 1.6244 1.4887 Ser-Val 1.4527 1.3741 1.3880 1.4709 1.28421.4622 1.3133 Met-Pro 0.9162 0.8752 1.0029 0.9704 0.9040 0.9563 1.0338Phe-Glu 0.9204 0.9451 0.9806 1.0519 1.0452 1.0352 1.0354 Ser-Asn 1.30931.2165 1.0079 1.1002 1.2030 1.2715 1.2753 Ser-Gln 0.9996 1.2502 1.14531.2382 1.1929 1.2511 1.1504 Glu-Trp 0.9494 0.9011 0.9385 1.0170 0.93200.9492 1.0018 Trp-Leu 1.0357 0.9945 1.0236 1.0663 1.0234 1.0145 1.1681His-Trp 0.9439 0.9185 1.0126 1.0278 0.8973 1.0292 1.0811 **Aactinomycetemcomitans

TABLE 2 Beneficial bacteria at 24 hrs A naeslundi C sputigena S gordoniiA viscosus S salivarius S sanguinis V parvula S mitis Ser-Leu 1.49491.9645 1.5333 2.2523 1.4865 1.2625 1.4153 1.6052 Ser-Val 1.4073 1.45411.4328 2.1120 1.3826 1.2030 1.2776 1.4166 Met-Pro 1.0381 2.5077 1.08701.5865 0.9990 0.8599 0.7714 1.0020 Phe-Glu 0.9642 2.1428 0.8472 1.38241.0411 1.0204 0.8043 1.0644 Ser-Asn 1.3199 2.2418 0.8814 1.7174 1.19171.1935 1.1101 1.0807 Ser-Gln 1.2183 2.2409 0.9754 1.6173 1.1847 1.19010.8812 1.0595 Glu-Trp 1.1008 1.7818 1.1537 1.5845 1.0068 0.9983 0.83780.9457 Trp-Leu 1.1778 0.8565 1.2109 1.6496 1.0960 0.9310 0.8734 1.0445His-Trp 1.3153 1.3266 1.0431 1.8735 0.9767 1.0228 1.1364 0.9734

TABLE 3 Pathogenic bacteria at 48 hrs A actino** F nucleatu P gingivalisP intermedi T forsythia S mutans S sobrinus Ser-Leu 2.2912 1.7358 1.84401.8484 1.8466 2.2977 2.0704 Ser-Val 1.9697 1.5983 1.9267 1.7953 1.72602.0721 1.8354 Met-Pro 0.8616 0.8113 1.0774 0.9393 0.9403 0.9242 1.0538Phe-Glu 0.9345 0.9112 0.9029 0.9562 1.0015 0.9816 1.0539 Ser-Asn 1.72851.4289 1.2165 1.1329 1.4354 1.7588 1.7028 Ser-Gln 1.1574 1.6185 1.49991.3661 1.4647 1.7642 1.6280 Glu-Trp 0.8665 0.8364 0.8770 0.9148 0.93031.0170 0.9874 Trp-Leu 1.1455 1.0254 1.2127 1.0399 1.1079 1.1337 1.2761His-Trp 0.9818 0.9774 1.3518 1.2057 0.9897 1.5031 1.2074 **Aactinomycetemcomitans

TABLE 4 Beneficial bacteria at 48 hrs A naeslundi C sputigena S gordoniiA viscosus S salivarius S sanguinis V parvula S mitis Ser-Leu 1.88311.6522 1.8912 2.5540 2.0833 1.6049 1.4281 2.0461 Ser-Val 1.7338 1.49331.7640 2.4349 1.9626 1.5280 1.3095 1.8051 Met-Pro 1.0427 1.8376 1.04472.0588 0.9500 0.8295 0.6345 0.9214 Phe-Glu 0.9466 1.7139 0.9890 2.12240.9959 1.0208 0.7552 1.1176 Ser-Asn 1.6167 1.5699 1.3337 2.0421 1.58861.5794 1.1143 1.5785 Ser-Gln 1.5857 1.6963 1.4584 2.0308 1.6185 1.62980.8706 1.5980 Glu-Trp 1.2040 1.4150 1.4320 2.1016 0.9525 1.0208 0.78730.9396 Trp-Leu 1.4354 0.9454 1.2858 2.0774 1.1505 0.9561 0.7918 1.0524His-Trp 1.5439 1.3068 1.1070 2.2797 1.0689 1.0830 1.4269 1.0242

In the above data, a value of 2 was taken as the threshold above whichthe dipeptide caused markedly increased metabolic activity of thebacterium. This value was selected in order to exclude low-stimulatingmetabolites and avoid false positive results.

As can be seen from the above data, Ser-Leu, Ser-Val, Met-Pro, Phe-Glu,Ser-Asn and Ser-Gln exhibited prebiotic effects at 24 hrs, and Met-Pro,Phe-Glu, Ser-Asn, Ser-Gln, Glu-Trp, Trp-Leu and His-Trp exhibitedprebiotic effects at 48 hrs. Without being bound by any theory, it isbelieved that those substrates which exhibit prebiotic effects at 48 hrsmay provide beneficial effects to the oral cavity upon prolonged use.

As Ser-Leu and Ser-Val are metabolized faster by the beneficial bacteriathan by the pathogenic bacteria (shown by a value of 2 in the above datasets being reached at 24 hrs for the beneficial bacteria but not for thepathogenic bacteria), the presence of Ser-Leu or Ser-Val would beexpected to cause the beneficial bacteria to suppress the growth of thepathogenic bacteria within a short timescale, thus maintaining a healthyoral microbiota. As Ser-Leu and Ser-Val are metabolized faster by thebeneficial bacteria than by the pathogenic bacteria, these dipeptidesare consumed/metabolized (and thus taken out of the environment) by thebeneficial bacteria before the pathogenic bacteria can start using them.Without being bound by any theory, it is believed that the stimulatoryeffect of Ser-Leu and Ser-Val on the pathogenic bacteria as seen at 48hrs might be abolished when a mixture of beneficial and pathogenicbacteria is present (such as in the oral cavity), as the dipeptidesubstrates have already been metabolized by the beneficial bacteria andare therefore unavailable for use by the pathogenic bacteria. Thus, ifthe growth/metabolic activity/colonization of the beneficial bacteria isstimulated before that of the pathogenic bacteria, then the beneficialbacteria can multiply and exert an inhibitory effect on the pathogenicbacteria before the latter have the chance to grow/multiply.

Example 2

The extent of growth of various beneficial oral and pathogenic oralbacteria in the presence of the dipeptides Met-Pro and Phe-Glu, asexamples, was investigated. The amino acid residues present in thedipeptides are L-amino acid residues.

The beneficial oral bacteria tested were Streptococcus mitis,Streptococcus salivarius, Streptococcus oralis, Actinomyces viscosus,Streptococcus gordonii, Capnocytophaga sputigena, Actinomyces naeslundiiand Veillonella parvula. The pathogenic oral bacteria tested wereStreptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis,Fusobacterium nucleatum, Tannerella forsythia, Aggregatibacteractinomycetemcomitans and Streptococcus sobrinus.

Substrates that were able to increase the growth (or maximal growthdensity or extent of growth) of at least one of the above beneficialbacteria while not or only minimally increasing the growth (or maximalgrowth density) of the pathogenic bacteria are considered to beprebiotic compounds.

The extent of growth of the tested bacteria in response to selectedcompounds (Met-Pro, Phe-Glu) was investigated by setting up growthcurves in a nutritionally rich medium (brain heart infusion broth (BHI),Oxoid), over 48 hours. Late exponential growth phase liquid cultureswere prepared by transferring the respective bacterium from blood agarplates to BHI and overnight incubation at 37° C. in an anaerobicatmosphere for A. viscosus, V. parvula, F. nucleatum, P. gingivalis, P.intermedia, T forsythia, A. naeslundii, and C. sputigena, and in a 5%CO₂ environment for S. salivarius, S. sanguinis, S. mitis, A.actinomycetemcomitans, S. mutans, S. sobrinus and S. gordonii. Overnightcultures were transferred to BHI and adjusted to a concentration of1×10⁷ CFU/ml (colony forming units per ml) by measuring the opticaldensity at 600 nm (OD₆₀₀) (BioRad SmartSpec 3000). For each strain, 200μl of the bacterial suspension was added to a 96-well plate containing20μl of the respective di-peptides. Final concentrations of thedi-peptides were set to 5, 10, 20 and 25 μmol/ml. For each bacteriumtested, a respective control was also used, which did not contain thedipeptide. Plates were incubated as previously described.

For each combination of bacterium/dipeptide, and for each of theirrespective controls (i.e. the particular bacterium in the nutritionallyrich medium with no dipeptide), the optical density was measured at 630nm (OD₆₃₀) at 0 h, 24 h and 48 h (Thermo Scientific Multiskan Ascent).Additionally, the OD₆₃₀ for combinations of bacterium/dipeptide grown ina 5% CO₂ environment were also measured every hour between 0 h and 9 h.For each combination of bacterium/dipeptide, the maximal OD valueobtained over the 48 hr time period was divided by the maximal OD valueobtained for the respective control over the 48 hr period, so that thecontrol had a value of 1. A value greater than 1 for a particularcombination of bacterium with dipeptide therefore indicates that theextent of bacterial growth over 48 hrs was greater than extent ofbacterial growth over 48 hrs for the control.

The experiment was carried out on three different days (thus providing 3biological replicas) and on each day the experiment was carried out inquadruple (thus providing 4 technical replicas) for each combination ofbacterium with dipeptide and for each control. For each day and for eachcombination, the average (mean) of the values obtained (as detailedabove) for the four technical replicas was calculated to provide asingle value for each combination on each day. The values shown inTables 5 to 6, below, are the average (mean) of the three single valuesobtained for each combination of bacterium with dipeptide. The resultsare shown in Tables 5 to 6, below:

TABLE 5 Pathogenic bacteria over 48 hours Phe-Glu Phe-Glu Phe-GluPhe-Glu Met-Pro Met-Pro Met-Pro Met-Pro 25 μmol/ml 20 μmol/ml 10 μmol/ml5 μmol/ml 25 μmol/ml 20 μmol/ml 10 μmol/ml 5 μmol/ml A actino* 0.23200.2201 0.3227 0.5643 0.2297 0.3282 0.6321 0.7454 F nucleatum 0.24020.2311 0.8493 0.7990 0.7273 0.7782 0.8667 0.8422 P gingivalis 0.42080.4181 0.9810 0.8526 0.4276 0.7021 0.8266 0.8702 P intermedia 0.22240.2094 0.2856 1.1149 0.2336 0.3193 0.9742 0.9177 S mutans 0.2492 0.42120.9003 0.9703 0.5618 0.7929 0.9083 0.9473 S sobrinus 0.4315 0.58320.9900 1.0790 0.8547 1.0167 1.0301 0.9294 T forsythia 0.8302 0.77070.7490 1.0118 0.7018 0.7533 0.9743 0.9244 *A actinomycetemcomitans

TABLE 6 Beneficial bacteria over 48 hours Phe-Glu Phe-Glu Phe-GluPhe-Glu Met-Pro Met-Pro Met-Pro Met-Pro 25 μmol/ml 20 μmol/ml 10 μmol/ml5 μmol/ml 25 μmol/ml 20 μmol/ml 10 μmol/ml 5 μmol/ml A naeslundii 0.16530.1648 0.4962 0.7791 0.3360 0.5466 0.8131 0.8420 C sputigena 1.18041.0801 0.9872 0.9921 0.9701 2.9796 3.5579 0.9618 S gordonii 0.29080.3992 0.8613 0.9682 0.6853 0.7735 0.9528 0.9757 A viscosus 0.15630.1598 0.3845 0.5112 0.2498 0.3154 0.4102 0.4873 S salivarius 0.23220.2953 0.8123 0.9916 0.5262 0.7572 1.0153 0.9729 S sanguinis 0.23340.2451 0.5644 0.8140 0.5011 0.6852 0.9070 0.9059 V parvula 1.0475 0.95331.0199 1.0286 0.9671 0.9516 0.9808 0.9980 S mitis 0.4367 0.3250 2.06421.9957 1.7501 1.8922 1.2332 0.9134

In the above data, a value of 1.25 was taken as the threshold abovewhich the dipeptide caused markedly increased extent of growth of thebacterium relative to the control. This value was selected in order toexclude low-stimulating metabolites and avoid false positive results.

As can be seen from the above data, Phe-Glu, exhibited prebiotic effectsat concentrations of 5 and 10 μmol/ml, and Met-Pro, exhibited prebioticeffects at concentrations of 25, 20 and 10 μmol/ml.

Example 3

The effects of the dipeptides Met-Pro and Phe-Glu, as examples, uponbiofilm growth of various beneficial oral bacteria and pathogenic oralbacteria was also investigated. The amino acid residues present in thedipeptides are L-amino acid residues.

Substrates that were able to increase the biofilm mass of at least oneof the above beneficial bacteria while not or only minimally increasingthe biofilm mass of the pathogenic bacteria are considered to beprebiotic compounds.

The increase in biofilm formation of the tested bacteria in response toselected compounds (Met-Pro, Phe-Glu) was investigated by setting upbiofilm growth assays in a nutritionally rich medium (brain heartinfusion broth (BHI), Oxoid), over 48 hours. Late exponential growthphase liquid cultures were prepared by transferring the respectivebacterium from blood agar plates to BHI and overnight incubation at 37°C. in an anaerobic atmosphere for A. viscosus, V. parvula, F. nucleatum,P. gingivalis, P. intermedia, T forsythia, A. naeslundii, and C.sputigena, and in a 5% CO₂ environment for S. salivarius, S. sanguinis,S. mitis, A. actinomycetemcomitans, S. mutans, and S. gordonii.Overnight cultures were transferred to BHI and adjusted to aconcentration of 1×10⁷ CFU/ml by measuring the optical density at 600 nm(OD₆₀₀) (BioRad SmartSpec 3000). For each strain, 200 μl of thebacterial suspension was added to a 96-well plate containing 20 μl ofthe respective di-peptides. Final concentrations of the di-peptides wereset to 5, 10, 20 and 25 μmol/ml. For each bacterium tested, a respectivecontrol was also used, which did not contain the dipeptide.Additionally, for each bacterium tested, a background control, tocorrect for background staining, was added. This background controlcontained the bacterium tested and 0.03 weight % chlorhexidine, as anantiseptic. Plates were incubated as previously described. After 48 hrsthe supernatant was removed from the wells of the plates. The wells werewashed twice with 1×100 μL PBS (phosphate buffered saline), fixed for 20minutes with 96% vol. ethanol (96% vol. solution in water) and thebiofilm retained at the bottom of the wells was stained with 1 weight %crystal violet (1 weight % solution in water). The bound dye wasdissolved with 5%vol. acetic acid (5% vol. solution in water).Quantification of the stained biofilm was performed by measuring theabsorbance at 630 nm using a Multiskan Ascent microplate reader (ThermoScientific).

For each combination of bacterium/dipeptide, the OD value obtained at 48hrs was divided by the OD value obtained for the respective controlafter subtracting the OD value of the background control, so that thecontrol had a value of 1. A value greater than 1 for a particularcombination of bacterium with dipeptide therefore indicates that thebiofilm growth over 48 hrs was greater than the biofilm growth over 48hrs for the control.

The experiment was carried out on three different days (thus providing 3biological replicas) and each day the experiment was carried out inquadruple (thus providing 4 technical replicas) for each combination ofbacterium with dipeptide and for each control. For each day and for eachcombination, the average (mean) of the values obtained (as detailedabove) for the four technical replicas was calculated to provide asingle value for each combination on each day. The values shown inTables 7 to 8, below, are the average (mean) of the three single valuesobtained for each combination of bacterium with dipeptide. The resultsare shown in Tables 7 to 8, below:

TABLE 7 Pathogenic bacteria at 48 hours Met-Pro Met-Pro Met-Pro Met-ProPhe-Glu Phe-Glu Phe-Glu Phe-Glu 25 μmol/ml 20 μmol/ml 10 μmol/ml 5μmol/ml 25 μmol/ml 20 μmol/ml 10 μmol/ml 5 μmol/ml A actino* 0.11030.2556 0.5095 0.8177 0.0212 0.0481 0.3454 0.5867 F nucleatum 0.52160.6796 0.5568 0.6087 0.0112 0.0056 0.2232 0.2553 P gingivalis 0.49900.8922 0.7344 0.6831 0.3008 0.1717 0.9004 0.7552 P intermedia 0.11480.2095 0.9773 1.2520 0.2516 0.2103 0.1852 0.7904 S mutans 1.3235 0.96250.0118 0.0252 0.4072 1.2009 0.5693 0.0781 T forsythia 0.1909 0.27260.4691 0.4857 0.0254 0.0188 0.3083 0.5744 *A actinomycetemcomitans

TABLE 8 Beneficial bacteria over 48 hours Met-Pro Met-Pro Met-ProMet-Pro Phe-Glu Phe-Glu Phe-Glu Phe-Glu 25 μmol/ml 20 μmol/ml 10 μmol/ml5 μmol/ml 25 μmol/ml 20 μmol/ml 10 μmol/ml 5 μmol/ml A naeslundii 0.12810.4130 0.9986 1.1703 0.0067 0.0174 0.4234 0.9914 C sputigena 0.01380.1660 0.1949 0.2411 0.2648 0.1578 0.1079 0.1909 S gordonii 1.43721.6395 1.8572 1.4212 0.2237 0.6462 1.7019 1.8315 A viscosus 0.03550.1766 0.4021 0.4286 0.0490 0.0684 1.2242 1.3972 S salivarius 1.47341.0385 0.9847 0.8003 0.7342 0.8978 1.0359 1.4007 S sanguinis 1.65802.1767 1.7015 1.1768 0.4820 0.5417 1.0551 1.2254 V parvula 0.2121 0.17450.2886 0.2845 0.2969 0.1638 0.3136 0.3257 S mitis 0.0464 0.2959 1.40081.3974 0.1018 0.0264 0.0179 0.7237

In the above data, a value of 1.6 was taken as the threshold above whichthe dipeptide caused markedly increased biofilm growth of the bacteriumrelative to the control. This value was selected in order to excludelow-stimulating metabolites and avoid false positive results.

As can be seen from the above data, Phe-Glu, exhibited prebiotic effectsat concentrations of 5 and 10 μmol/ml, and Met-Pro, exhibited prebioticeffects at concentrations of 25, 20 and 10 μmol/ml.

1. An oral care composition comprising: (a) a dipeptide of the formulaXaa1-Xaa2 or Xaa2-Xaa1; wherein Xaa1 is an amino acid with a polaruncharged side chain; and Xaa2 is selected from an amino acid with ahydrophobic side chain, an amino acid with a polar uncharged side chain,and proline; or (b) a dipeptide of the formula Xaa3-Xaa4 or Xaa4-Xaa3;wherein Xaa3 is an amino acid with a hydrophobic side chain; and Xaa4 isselected from an amino acid with a hydrophobic side chain and an aminoacid with a charged side chain.
 2. The oral care composition of claim 1,wherein Xaa1, Xaa2, Xaa3 and Xaa4 are L-amino acids.
 3. The oral carecomposition of claim 1, wherein the dipeptide is present in thecomposition in an amount of from 0.01 weight % to 10 weight %, based onthe weight of the oral care composition.
 4. The oral care composition ofclaim 1, wherein Xaa2 is an amino acid with an aliphatic hydrophobicside chain.
 5. The oral care composition of claim 4, wherein Xaa2 isalanine, isoleucine, leucine or valine.
 6. The oral care composition ofclaim 5, wherein Xaa2 is leucine or valine.
 7. The oral care compositionof claim 1, wherein Xaa2 is asparagine, cysteine, glutamine, methionine,serine or threonine.
 8. The oral care composition of claim 7, whereinXaa2 is asparagine or glutamine.
 9. The oral care composition of claim1, wherein Xaa2 is proline.
 10. The oral care composition of claim 1,wherein Xaa1 is serine, methionine, threonine, cysteine, asparagine orglutamine.
 11. The oral care composition of claim 10, wherein Xaa1 isserine.
 12. The oral care composition of claim 10, wherein Xaa1 ismethionine.
 13. The oral care composition of claim 1, wherein Xaa3 is anamino acid with an aromatic hydrophobic side chain.
 14. The oral carecomposition of claim 13, wherein Xaa3 is tryptophan, phenylalanine, ortyrosine.
 15. The oral care composition of claim 14, wherein Xaa3 istryptophan.
 16. The oral care composition of claim 14, wherein Xaa3 isphenylalanine.
 17. The oral care composition of claim 13, wherein Xaa4is aspartic acid, glutamic acid, arginine, histidine or lysine.
 18. Theoral care composition of claim 15, wherein Xaa4 is glutamic acid orhistidine.
 19. The oral care composition of claim 13, wherein Xaa4 is anamino acid with an aliphatic hydrophobic side chain.
 20. The oral carecomposition of claim 19, wherein Xaa4 is alanine, isoleucine, leucine orvaline.
 21. The oral care composition of claim 1, wherein thecomposition is a dentifrice, a toothpaste, a gel, a tooth powder, amouthwash, a mouthrinse, a lozenge, a tablet, a spray, a gum, or a film.22. The oral care composition of claim 1, wherein the compositionfurther comprises at least one species of bacteria that has beneficialeffects on oral health.
 23. The oral care composition of claim 22,wherein the species of bacteria that has beneficial effects on oralhealth is selected from Streptococcus mitis, Streptococcus salivarius,Streptococcus sanguinis, Actinomyces viscosus, Veillonella parvula,Streptococcus gordonii, Capnocytophaga sputigena, Actinomyces naeslundiiand combinations thereof.
 24. A composition according to claim 1, foruse in selectively promoting, in an oral cavity: growth, metabolicactivity or colonization of bacteria that have beneficial effects onoral health, relative to growth, metabolic activity or colonization ofpathogenic oral bacteria.
 25. A composition according to claim 1, foruse in selectively promoting, in an oral cavity, biofilm formation bybacteria that have beneficial effects on oral health, relative tobiofilm formation by pathogenic oral bacteria.
 26. A compositionaccording to claim 1, for use in maintaining and/or re-establishing ahealthy oral microbiota.
 27. A composition according to claim 1, for usein preventing one or more of gingivitis, periodontitis,peri-implantitis, peri-implant mucositis, necrotizing gingivitis,necrotizing periodontitis and caries.
 28. A method of selectivelypromoting, in an oral cavity of a subject: growth, metabolic activity orcolonization of bacteria that have beneficial effects on oral health,relative to growth, metabolic activity or colonization of pathogenicoral bacteria; the method comprising contacting the oral cavity with anoral care composition of claim
 1. 29. A method of selectively promoting,in an oral cavity of a subject, biofilm formation by bacteria that havebeneficial effects on oral health, relative to biofilm formation bypathogenic oral bacteria; the method comprising contacting the oralcavity with an oral care composition of claim
 1. 30. A method ofmaintaining and/or re-establishing a healthy oral microbiota in asubject, the method comprising contacting an oral cavity of the subjectwith an oral care composition of claim
 1. 31. A method of preventing oneor more of gingivitis, periodontitis, peri-implantitis, peri-implantmucositis, necrotizing gingivitis, necrotizing periodontitis and cariesin a subject, the method comprising contacting an oral cavity of thesubject with an oral care composition of claim
 1. 32-34. (canceled)